NMR-Guided Isolation of Anti-inflammatory Carabranolides from the Fruits of Carpesium abrotanoides L.

Carabranolides present characteristic NMR resonances for the cyclopropane moiety, which distinctly differ from those of other compounds and were used for an NMR-guided isolation in this study. As a result, 11 undescribed carabranolides (1–11), along with five known ones (12–16), were isolated from the fruits of Carpesium abrotanoides L. Compounds 1–11 are new esters of carabrol at C-4 with different carboxylic acids. Their structures were elucidated by HRESIMS and NMR spectroscopic data analysis. The biological evaluation showed that compounds 2–4, 15, and 16 exhibited significant inhibitory activity against LPS-induced NO release with an IC50 value of 5.6–9.1 μM and dose-dependently decreased iNOS protein expression in RAW264.7 cells.

T he genus Carpesium (Compositae family) comprises about 21 species, which are widely distributed in Asia and Europe, particularly in the mountainous areas of Southwest China. 1 In China, 17 species and three varieties are known, and six species (Carpesium abrotanoides L., C. cernuum L., C. divaricatum Sieb.et Zucc., C. lipskyi Winkl., C. macrocephalum Franch.et Sav., and C. nepalense Less) have been used as traditional Chinese medicines to treat cold, fever, sore throat, tonsillitis, wound bleeding, swollen poison, herpes zoster, and snake bites. 2,3Sesquiterpene lactones and sesquiterpene dimers were reported as characteristic constituents of this genus with diverse biological activities including cytotoxic, 4 anti-inflammatory, 5 antibacterial, 6 and insecticidal 7 activities.
−10 Fruits of this species serve as a significant anthelmintic in traditional Chinese medicine for ascariasis, pinworm disease, and tapeworm infections. 11lthough over 130 compounds have been identified from C. abrotanoides, 12−14 the bioactive compounds from its fruits have rarely been investigated. 7,13In a recent study, seven new cytotoxic sesquiterpene lactone dimers with a carabrol unit were isolated from the fruits of C. abrotanoides. 13Our preliminary NMR data analysis revealed that a number of unknown carabrol derivatives were present in the extract of the fruits of C. abrotanoides.To identify these unknown carabranolides and their bioactivities, an NMR-guided isolation was performed on the fruits of C. abrotanoides, which led to the isolation of 11 new carabranolides (1−11), along with five known derivatives (12−16).The anti-inflammatory activity of these sesquiterpenes was also evaluated, revealing that compounds 2−4, 15, and 16 significantly inhibited LPSinduced NO release with an IC 50 value of 5.6−9.1 μM and dose-dependently decreased iNOS protein expression in RAW264.7 cells.
Compound 2 exhibited the same molecular formula as 1 by (+)-HRESIMS analysis at m/z 477.2838.The IR and NMR spectra of 2 were very similar to those of 1, indicating that 2 was also a carabrol derivative.A comparison of the 13 C NMR data (Table 1) of 2 with those of 1 revealed that these compounds were nearly identical, except that the resonances of C-8′, C-9′, and C-10′ of 2 shifted downfield in a range of 2−5 ppm compared with those of 1, which could be due to the configurational change at C-9′ in the furanoctanoic acid unit of 2. The J 9′,10′ coupling constant of 6.7 Hz supported that H-9′ and H-10′ were opposite, the same as in lonfuranacid B. 20 Therefore, the structure of 2 was determined as lonfuranacid B-4-O-carabrol, and it was named carabrolate B.
Compound 4 was isolated as a yellow oil.HRESIMS analysis of 4 gave a [M + H] + peak at m/z 399.216 (calcd for 399.2166), indicating the molecular formula C 24 H 30 O 5 , with 10 degrees of unsaturation.The presence of a carabrol group in 4 was supported by an analysis of 1 H and 13 C NMR data (Tables 1 and 2).In addition to resonances for carabrol, an ABX aromatic system δ H 6.61 (dd, J = 8.2, 2. 7

Journal of Natural Products
The HMBC correlations from H-2′ to C-1′, C-4′, and C-8′, H-9′ to C-3′ and C-5′, and H-4 to C-1′ (Figure 2) were further indicative of a 2-methyl-4-hydroxyphenylacetic acid 22 unit in 4, attached to C-4 of the carabraol group.Compared to those of 3, chemical shifts of H-1−6, H-9, H-14, and H-15 of 4 were upfield in a range of 0.04−0.33ppm, which could be caused by the shielding effect, inductive effect, and spatial proximity of the 2-methyl-4-hydroxyphenylacetic acid moiety in 4. Thus, the structure of 4 was determined as shown and named carabrolate D.
The oxylipin group in 5 exhibited almost identical NMR data to those of a natural 6,9,10-trihydroxyoctadec-7-enoic acid, 23 which were confirmed by 2D NMR analysis (Figure 2).The coupling constant of H-9′, 10′ (J 9′,10′ = 6.2 Hz) and identical NMR data to those of 9R,10R-trihydroxyoctadec-7-enoic acid 23 deduced the 9′β-OH, and 10′β-OH in 5. Thus, the structure of 5 was determined as trihydroxyoctadec-7-enate. 1D and 2D NMR data analysis revealed that 5 and 6 have the same planar structure.The major differences between them were the chemical shifts of C-5′ to C-8′ and the NOE correlation of H-6′/H-8′ in 5 but not in 6, which suggested that the hydroxy group at the C-6′ in 6 was opposite to that of 5. 24 To establish the absolute configurations of the C-6′, C-9′, and C-10′ in 5 and 6, vicinal diols in 5 and 6 were first protected by ketals to give 5a and 6a (Figure 3) according to the previously described procedure. 25The secondary alcohol at C-6′ in 5a and 6a was then derivatized with (S)-and (R)-αmethoxy-α-(trifluoromethyl)phenylacetyl (MTPA) chlorides, 26,27 yielding their S-and R-MTPA esters (5b/5c, 6b/ 6c) (Figure 3).By analysis of Δδ S−R values of the 1 H NMR chemical shift between 5b and 5c and between 6b and 6c,    respectively, the absolute configurations of the C-6′ in 5 and 6 were designated as S and R, respectively (Figure 3).Based on the relative configuration of C-9′ and C-10′, the absolute configurations of oxylipin moieties in 5 and 6 were determined as 6′S,9′R,10′R and 6′R,9′R,10′R, respectively.1 and 3), except for varying degrees of oxidation.The fatty acid in 7 was established as 10,16-dihydroxy hexadecanoic acid by the observation of an oxygenated methylene at δ H 3.65 (t, J = 6.6 Hz, H-16′), an oxygenated methine δ H 3.58 (dq, J = 8.4, 5.1 Hz, H-10′), and 13 methylenes at δ H 1.30−2.26(m), as well as 16 carbon signals including 14 methylenes (an oxygenated methylene at δ C 63.0, C-16′), an oxygenated methine at δ C 72.0, C-10, and a quaternary carbon at δ C 173.5 (C-1′) in the 1 H and 13 C NMR spectra of 7, which were consistent with the data of 10,16-dihydroxy hexadecanoic acid. 28The position of the hydroxy group at C-10′ in 7 was further confirmed by characteristic fragment ions at m/z 271.2225, 187.1491, and 169.1209 in its HRESIMS/MS spectrum (Figure 4).By comparison with known spectroscopic data, the fatty acid groups of 8 and 9 were verified to be 9-hydroxynonanoic acid 29 and 3hydroxyheptadecanoic acid, respectively. 30The HMBC correlations from H-9′ to C-7′/C-8′ in 8 and from H-3′ to C-1′/C-2′/4′ in 8 also supported the existence of 9′-OH in 8 and 3′-OH in 9 (Figure 2).The secondary alcohol at C-3′ in 9 was derivatized with (S)-and (R)-MTPA chloride, yielding its S-and R-MTPA esters (9a and 9b) (Figure 5).The absolute configuration of C-3′ in 9 was determined as S based on the Δδ S−R values of its Mosher esters (9a and 9b).Moreover, for compounds 7−9, fatty acid groups attaching to C-4 of carabrol were established by their key HMBC correlations between H-4 (δ H 4.91−4.96)and C-1′ (δ C 172.7−173.5)(Figure 2).Hence, the structures of 7−9 were determined as shown and named carabrolates G (7) for 421.2585).The 1 H and 13 C NMR spectroscopic data (Tables 1 and 3) of 10 showed the presence of the carabrol moiety.Moreover, the resonances of an azelaic acid group in 10 were revealed by the seven methylenes (δ H 1.31−2.32and δ C 24.7−34.7)and two carbonyl carbons (δ C 173.5, C-1′ and 179.0,C-9′) in the 1 H and 13 C NMR spectra.Furthermore, these data were identical to those of the natural azelaic acid. 31he HMBC cross-peak between H-4 (δ H 4.91) and C-1′ (δ C 173.5) proved the connection of the carabranolide moiety and azelaic acid (Figure 2).Thus, the structure of 10 was determined as shown and named carabrolate J. Compound 11, a yellow oil, presented the molecular formula C 33 H 54 O 4 based on analysis of (+)-HRESIMS at m/z 537.391 [M + H] + (calcd for 537.3914).Its 1 H and 13 C NMR spectroscopic data (Tables 1 and 3) were extremely close to those of carabrol-4-O-linoleate (12) 17 in CDCl 3 , except for the absence of signals for a double-bond group at C-12′ and C-13′.Compound 11 was comprised of a carabrol unit and oleic acid group through HRMS and NMR data analysis. 32The position of the double bond at C-9′ and C-10′ in 11 was verified by fragment ions at m/z 155.0859 and 143.0819 through HRESIMS/MS analysis (Figure 4).Moreover, the oleic acid group attaching to C-4 of carabrol was defined by the key HMBC cross-peak (Figure 2) between H-4/C-1′.Therefore, compound 11 was established as carabrolate K.
Given the anti-inflammatory activity of carabrol, 33 the activity of carabrol derivatives (1−16) on LPS-induced NO production in RAW264.7 cells was investigated.As shown in Table 4, compounds 1−8, 9, and 14−16 exhibited notable inhibitory effects on LPS-induced NO release in RAW264.7 cells, with IC 50 values ranging from 5.8 to 15.1 μM.All tested compounds had no obvious effect on cell viability at a concentration of 20 μM.Structurally, the principal distinction among compounds 1−16 is the substituents at the C-4 position.The unsubstituted carabrol (14) exhibited considerable inhibitory activity against NO production with an IC 50 value of 10.96 μM.When the 4-OH of carabrol was oxidized (15), acetylated (16), or esterified with 2-methyl-4-hydroxyphenylacetic acid (4), the IC 50 values of 4 (5.82 μM), 15 (6.77 μM), and 16 (5.64μM) decreased almost one-fold compared with carabrol.In contrast, the esterification of carabrol with the long-chain fatty acids diminished the bioactivity, while cyclic or unsaturated and oxidized fatty    4).

■ EXPERIMENTAL SECTION
General Experimental Procedures.Optical rotations were defined by an Autopol I polarimeter (Rudolph, Flanders, USA).UV and ECD spectra were recorded with a J-1500 circular dichroism spectrometer (JASCO, Japan).IR data were measured with an IR Affinity-1S spectrometer (Shimadzu, Japan).NMR experiments were recorded with a Bruker Ascend-600 spectrometer (Bruker, Germany) with TMS used as an internal standard.An Agilent-6230 Q-TOF mass spectrometer with an Agilent 1260 UHPLC system was used to obtain HRESIMS data (Agilent, USA).HRESIMS/MS analyses for 7 and 11 were performed on an Agilent-6230 Q-TOF with the energy variable from 10 to 40 eV.Medium-pressure liquid chromatography (MPLC) was performed on a Buchi C-620 system (Buchi, Switzerland) equipped with a Siliabond C 18 column (ODS gel, 5 μm, 49 × 460 mm) with a flow rate of 100 mL/min.Semipreparative HPLC was performed on an Agilent 1260 HPLC system (Agilent, USA) coupled with a DAD detector by using an XBrdige C 18 column     (Cleveland, OH, USA).CDCl 3 containing TMS (1%) was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA).Deionized water was obtained using a Millipore Milli-Q-plus system (Millipore, Bedford, MA, USA).
Carabrolate Ketalization for Compounds 5 and 6.Treatment of 5 and 6 (1.0 mg) with PDSA (30 μg) in 400 μL of acetone afforded the acetone ketal at 37 °C for 2 h.The reaction was monitored by UHPLC-QTOF-MS analysis.Then the mixtures were partitioned with CHCl 3 and H 2 O to give 5a and 6a.
Preparation of (S)-and (R)-MTPA Esters of 5a, 6a, and 9. Compounds 5a, 6a, and 9 (0.5 mg) were dissolved in 500 μL of deuterated pyridine.Then, 1.0 mg of 4-dimethylaminopyridine and 5 μL of (S)-or (R)-MTPA chlorides were added to the above solutions, which were allowed to react at room temperature for 2 h.The mixtures were subjected to an NMR spectrometer, and their 1 H and 1 H− 1 H COSY NMR spectra were recorded.
Cell Lines and Cell Culture.The RAW264.7 cell line was obtained from ATCC (USA) and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) (Gibco, UK) in a humidified atmosphere with 5% CO 2 at 37 °C.
Assay for NO Inhibitory Activity.NO inhibitory activity of selected compounds was evaluated by using an LPS-induced cell model.Briefly, RAW264.7 cells were inoculated into 96-well plates (1 × 10 5 cells/well) and were treated with the test compounds or dexamethasone (DEX, a positive control) at concentrations of 1.25, 2.5, 5, 10, and 20 μM in triplicate.After being induced with LPS (100 ng/mL) for 24 h, the cell culture supernatants were collected to measure levels of NO by using the Griess reagent (Beyotime Biotechnology, People's Republic of China) according to the manufacturer's instructions.Finally, the absorbance at 550 nm was measured with a microplate reader.Concurrently, the MTT assay was employed to assess the viability of RAW264.7 cells, thereby evaluating the cytotoxic effects of the compounds under investigation. 34,35estern Blot Analysis.In brief, RAW264.7 cells were seeded in six-well plates at a density of 5 × 10 5 cells/well and treated with various concentrations (10, 20, and 40 μM) of compounds 2−4, 15, and 16 or DEX (5 μM), followed by stimulation with LPS (0.5 μg/ mL) for 24 h.Subsequently, cells were collected and lysed by using RIPA buffer.The extracted proteins were then separated by 8% SDS-PAGE and transferred onto PVDF membranes.These membranes were incubated overnight at 4 °C with primary antibodies of iNOS and GAPDH.After washing thrice with TBST, the membranes were exposed to a mouse monoclonal IgG conjugated secondary antibody for 1 h at room temperature.The protein bands were visualized by the LI-COR Odyssey imaging system (Lincoln, NE, USA).
Statistical Analysis.In the graph, data are presented as mean ± SD.The one-way ANOVA was used to assess the differences among the groups.Analysis of data was derived from the results of GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA).Values of p < 0.05 were considered to indicate statistical significance.

Figure 1 .
Figure 1. 1 H (A) and 13 C (B) NMR spectra of the PE fraction of the EtOH extract, the target fraction, a target compound, and carabrol (14) from the fruits of C. abrotanoides.
923H058) was purchased from Solarbio (China).LPS was purchased from Sigma-Aldrich (St. Louis, MO, USA).Plant Material.The fruits of C. abrotanoides were purchased in May 2022 from the Bozhou medicinal materials market in Anhui Province, People's Republic of China, and identified by Dr. Guo-yuan Zhu from Macau University of Science and Technology.The voucher specimen (CA-2022-05) is already stored in the State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology.Extraction and Isolation.The dried fruits of C. abrotanoides (15 kg) were powdered and extracted with 80% EtOH at room temperature.The extract was concentrated under reduced pressure to afford 1.6 L of the concentrated extract, which was then partitioned successively with PE and EtOAc.The 1 H and 13 C NMR spectra of PE and EtOAc extracts demonstrated the characteristic signals indicative of carabranolide derivatives, which were used to guide the further isolation of targeted compounds.The PE extract (241g) was separated by silica gel CC (PE− EtOAc−MeOH, 1:0:0 to 0:1:1), and the eluates were analyzed by TLC and combined to give 6 fractions.NMR analyses of these

Table 1 .
Compounds 7−9 were obtained as a yellow oil, with the molecular formulas C 31 H 52 O 6 , C 24 H 38 O 5 , and C 32 H 54 O 5 , determined by analysis of the corresponding HRESIMS ions [M + Na] + at m/z 543.3656, 429.2608, and 541.3863, respectively.Compounds 7−9 are also esters of carabrol composed of fatty acids, as indicated by the analysis of NMR data (Tables

■ ASSOCIATED CONTENT * sı Supporting Information The
Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jnatprod.4c00338.NMR, HRESIMS, IR, and UV spectra of compounds 1− 11 (PDF) Li-Ping Bai − State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau SAR 999078, People's Republic of China; orcid.org/0000-0002-2806-6883;Email: lpbai@ must.edu.moZhi-Hong Jiang − State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau SAR 999078, People's Republic of China; orcid.org/0000-0002-7956-2481;Email: zhjiang@must.edu.moGuo-Yuan Zhu − State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau SAR 999078, People's Republic of China; orcid.org/0000-0002-4355-894X;Email: gyzhu@must.edu.moState Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau SAR 999078, People's Republic of China Can-Can Wang − State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau SAR 999078, People's Republic of China Wenyue Tian − State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau SAR 999078, People's Republic of China Zhiyan Liu − State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau SAR 999078, People's Republic of China Meng-Yu Bao − State Key Laboratory of Quality Research in AuthorsLu Fu − Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Journal of Natural Products